The modulation of the tolerance of oocytes to cryopreservation by artificial expression of water channels -vertebrates and invertebrates-

通过水通道的人工表达调节卵母细胞对冷冻保存的耐受性-脊椎动物和无脊椎动物-

基本信息

批准号：
16380190
负责人：
KASAI Magosaburo
金额：
$ 9.6万
依托单位：
Kochi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

The permeability of the plasma membrane to water and cryoprotectants is an important factor determining the tolerance of cells to cryopreservation and suitable condition for cryopreservation. There are two pathways for the movement of water and cryoprotectants through the plasma membrane, i.e., simple diffusion across the plasma membrane and facilitated diffusion via channel processes. In this study, we tried to clarify the relationship between the permeability of the plasma membrane and cryo-sensitivity in oocytes and embryos of various species.As a model for mammals, we used mice, and the permeability to water and cryoprotectants of oocytes and embryos at various stages were examined from the viewpoint of the expression of a water/cryoprotectant channel. It was found that in embryos at the morula stage, water and some cryoprotectants moved rapidly through the plasma membrane predominantly by facilitated diffusion via aquaporin 3 (AQP3), a water/cryoprotectant channel, whereas in oocy … More tes and embryos at early cleaved stages, water and cryoprotectants moved slowly through the plasma membrane predominantly by simple diffusion.In fish and amphibians, the cryopreservation of oocytes and embryos has not been realized. We tried to find suitable conditions for the cryopreservation of the oocytes from the viewpoint of membrane permeability. We used medaka and zebrafish as models for fish, and Xenopus laevis as a model for amphibians. In medaka and zebrafish, the permeability to water and cryoprotectants of the plasma membrane was higher in immature oocytes than mature oocytes. The high permeability of immature oocytes was increased further by the artificial expression of AQP3 by injecting AQP3 cRNA into them. Similar increase in the permeability to water and cryoprotectants was observed in AQP3 cRNA-injected immature Xenopus oocytes. However, considering the large volume of the oocyte in these animals, further increase in membrane-permeability would be necessary for successful cryopreservation of oocytes.In addition, we tried to cryopreserve insect embryos, derived from parthenogenetically activated oocytes of Nasonia vitripennis. Removal of the wax layer of Nasonia embryos was effective to make them permeable to water and cryoprotectants (ethylene glycol and DMSO). When wax-removed embryos were vitrified with ethylene glycol-based solutions, many embryos survived, and some (〜30%) of them developed to larvae. Therefore, the conservation of various strains of Nasonia vitripennis by the cryopreservation of embryos would become possible. Less

质膜对水和冷冻保护剂的渗透性是决定细胞对冷冻保存的耐受性和冷冻保存合适条件的重要因素。水和冷冻保护剂穿过质膜的运动有两种途径，即穿过血浆的简单扩散。在这项研究中，我们试图阐明质膜的渗透性与不同物种的卵母细胞和胚胎的低温敏感性之间的关系。我们使用小鼠作为哺乳动物模型，从水/冷冻保护剂通道表达的角度检查了各个阶段的卵母细胞和胚胎对水和冷冻保护剂的渗透性，发现在桑葚胚阶段的胚胎中，水和冷冻保护剂的渗透性。一些冷冻保护剂主要通过水通道蛋白 3 (AQP3)（一种水/冷冻保护剂通道）的促进扩散快速穿过质膜，而在卵细胞和早期胚胎中在卵裂阶段，水和冷冻保护剂主要通过简单扩散缓慢地穿过质膜。在鱼类和两栖动物中，卵母细胞和胚胎的冷冻保存尚未实现，我们试图从膜的角度寻找合适的卵母细胞冷冻保存条件。我们使用青鳉和斑马鱼作为鱼类模型，并使用非洲爪蟾作为两栖动物模型。未成熟卵母细胞的质膜对水和冷冻保护剂的渗透性高于成熟卵母细胞，通过将AQP3 cRNA注射到未成熟卵母细胞中，对水和冷冻保护剂的渗透性也有类似的增加。在注射 AQP3 cRNA 的未成熟非洲爪蟾卵母细胞中观察到，然而，考虑到这些动物中卵母细胞的体积较大，进一步增加。膜渗透性对于卵母细胞的成功冷冻保存是必要的。此外，我们尝试冷冻保存来自 Nasonia vitripennis 孤雌生殖激活的卵母细胞的昆虫胚胎，去除 Nasonia 胚胎的蜡层可以有效地使其对水和冷冻保护剂具有渗透性。当用乙二醇溶液玻璃化去蜡胚胎时，许多胚胎存活下来，有些胚胎存活下来。 (~30%) 发育成幼虫，因此，通过胚胎冷冻保存各种 Nasonia vitripennis 品系将成为可能。